Advances in spatial mass spectrometry enable in-depth neuropharmacodynamics
Authors: Sooraj Baijnath, Ibrahim Kaya, Anna Nilsson, Reza Shariatgorji, Per E. Andrén
Journal: Trends in Pharmacological Sciences (2022)

Mass spectrometry imaging (MSI) is a powerful analytical technique that combines the ability of microscopy to provide spatial information about multiple molecular species with the specificity of mass spectrometry for the unlabeled mapping of analytes in diverse biological tissues.MSI has been used for pharmacokinetic and pharmacodynamic studies, including drug transport and drug–drug interactions through the blood–brain barrier, which demonstrates its usefulness in neuropharmacological research.Recent advances have enabled improved detection of diverse functional biomolecules in the brain, including neurotransmitters, metabolites, neuropeptides, and lipids.MSI can be used to study effects of drugs on multiple biological pathways by monitoring regional changes in biomolecules. Mass spectrometry imaging (MSI) is a powerful technique that combines the ability of microscopy to provide spatial information about multiple molecular species with the specificity of mass spectrometry (MS) for unlabeled mapping of analytes in diverse biological tissues. Initial pharmacological applications focused on drug distributions in different organs, including the compartmentalized brain. However, recent technological advances in instrumentation, software, and chemical tools have allowed its use in quantitative spatial omics. It now enables visualization of distributions of diverse molecules at high lateral resolution in studies of the pharmacokinetic and neuropharmacodynamic effects of drugs on functional biomolecules. Therefore, it has become a versatile technique with a multitude of applications that have transformed neuropharmacological research and enabled research into brain physiology at unprecedented resolution, as described in this review. Mass spectrometry imaging (MSI) is a powerful technique that combines the ability of microscopy to provide spatial information about multiple molecular species with the specificity of mass spectrometry (MS) for unlabeled mapping of analytes in diverse biological tissues. Initial pharmacological applications focused on drug distributions in different organs, including the compartmentalized brain. However, recent technological advances in instrumentation, software, and chemical tools have allowed its use in quantitative spatial omics. It now enables visualization of distributions of diverse molecules at high lateral resolution in studies of the pharmacokinetic and neuropharmacodynamic effects of drugs on functional biomolecules. Therefore, it has become a versatile technique with a multitude of applications that have transformed neuropharmacological research and enabled research into brain physiology at unprecedented resolution, as described in this review. MSI is an analytical technique that has significantly improved approaches in drug research, pathological analysis, and studies of drug–target and drug–drug interactions [1.Goodwin R.J.A. et al.A critical and concise review of mass spectrometry applied to imaging in drug discovery.SLAS Discov. 2020; 25: 963-976Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 2.Nilsson A. et al.Mass spectrometry imaging in drug development.Anal. Chem. 2015; 87: 1437-1455Crossref PubMed Scopus (135) Google Scholar, 3.Norris J.L. Caprioli R.M. Analysis of tissue specimens by matrix-assisted laser desorption/ionization imaging mass spectrometry in biological and clinical research.Chem. Rev. 2013; 113: 2309-2342Crossref PubMed Scopus (505) Google Scholar, 4.Shariatgorji M. et al.Mass spectrometry imaging, an emerging technology in neuropsychopharmacology.Neuropsychopharmacology. 2014; 39: 34-49Crossref PubMed Scopus (72) Google Scholar]. MSI has advantages over other conventional imaging techniques since it combines the molecular specificity of MS with spatial histology and cytology, allowing simultaneous unlabeled tissue mapping of diverse molecules, ranging from small drugs and their metabolites to endogenous metabolites, lipids, peptides, and small proteins. Recent advances in MSI have facilitated the quantitative and simultaneous imaging of drugs and comprehensive neurotransmitter systems in brain tissue sections with high lateral resolution, which is not feasible with any other imaging technique. This innovative approach can greatly help efforts to understand tissue biodistribution and pharmacokinetic–pharmacodynamic relationships of drugs in early discovery phases, as well as their pharmacology, toxicology, and disease pathogenesis in the development phase. Therefore, MSI has accelerated pharmacokinetic–pharmacodynamic research [1.Goodwin R.J.A. et al.A critical and concise review of mass spectrometry applied to imaging in drug discovery.SLAS Discov. 2020; 25: 963-976Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar,2.Nilsson A. et al.Mass spectrometry imaging in drug development.Anal. Chem. 2015; 87: 1437-1455Crossref PubMed Scopus (135) Google Scholar]. In this review we focus on matrix-assisted laser desorption ionization (MALDI) (see Glossary) and desorption electrospray ionization (DESI), the two most commonly used surface ionization techniques in MSI, although we also consider use of secondary ion mass spectrometry (SIMS) ionization for subcellular MSI [5.Thomen A. et al.Subcellular mass spectrometry imaging and absolute quantitative analysis across organelles.ACS Nano. 2020; 14: 4316-4325Crossref PubMed Scopus (37) Google Scholar], and present some of the recent and potential applications of MSI in neuropharmacology. There have been numerous recent technological advances in MSI instruments and software. These advances have been reviewed in detail elsewhere [6.Buchberger A.R. et al.Mass spectrometry imaging: a review of emerging advancements and future insights.Anal. Chem. 2018; 90: 240-265Crossref PubMed Scopus (450) Google Scholar,7.Scupakova K. et al.Cellular resolution in clinical MALDI mass spectrometry imaging: the latest advancements and current challenges.Clin. Chem. Lab. Med. 2020; 58: 914-929Crossref PubMed Scopus (56) Google Scholar] and are beyond the scope of this review, which is intended to provide an overview and illustrations of current and potential MSI applications in neuropharmacology. However, this section briefly describes advantages bestowed by recent advances in MSI, particularly with the most widely used ionization techniques (Table 1, Key table): MALDI, followed by DESI (Figure 1) and SIMS.Table 1Key table. Summary of recent discoveries and potential applications enabled by MALDI- and DESI-MSI technological and methodological developments for spatial neuropharmacokinetics and neuropharmacodynamicsMSI technology and/or methodological developmentDiscoveries and potentialsRefsInstrumentationMALDI-MSI of tissues and cells at 1–5-μm lateral resolution.Cellular and subcellular imaging of lipid, metabolite, and peptide distributions in the brain. Study of drug effects on individual cell types in brain tissue sections.[8.Kompauer M. et al.Atmospheric pressure MALDI mass spectrometry imaging of tissues and cells at 1.4-μm lateral resolution.Nat. Methods. 2017; 14: 90-96Crossref PubMed Scopus (336) Google Scholar,79.Zavalin A. et al.Tissue protein imaging at 1 μm laser spot diameter for high spatial resolution and high imaging speed using transmission geometry MALDI TOF MS.Anal. Bioanal. Chem. 2015; 407: 2337-2342Crossref PubMed Scopus (131) Google Scholar]High-speed MALDI-MSI with a laser capable of pulse repetition rates up to 5 kHz and high digitizer acquisition rate (up to 50–150 pixels/s).Acquisition of high-resolution images from large sample areas. Enables high-throughput tissue imaging on practical time scales.[9.Bednarik A. et al.MALDI MS imaging at acquisition rates exceeding 100 pixels per second.J. Am. Soc. Mass Spectrom. 2019; 30: 289-298Crossref PubMed Scopus (15) Google Scholar,80.Ogrinc Potocnik N. et al.Use of advantageous, volatile matrices enabled by next-generation high-speed matrix-assisted laser desorption/ionization time-of-flight imaging employing a scanning laser beam.Rapid Commun. Mass Spectrom. 2015; 29: 2195-2203Crossref PubMed Scopus (105) Google Scholar,81.Prentice B.M. et al.High-speed MALDI MS/MS imaging mass spectrometry using continuous raster sampling.J. Mass Spectrom. 2015; 50: 703-710Crossref PubMed Scopus (68) Google Scholar]Transmission-mode MALDI-2 ionization source combined with high-mass resolving analyzer enabling highly sensitive detection of molecules that are difficult to ionize through ionization enhancement.Detection of low abundance and/or low-ionization efficiency drug molecules and drug metabolites at high spatial resolutions within single cells and tissue sections. The post-ionization technique reduces ion suppression effects and improves sensitivity in metabolomics.[14.Niehaus M. et al.Transmission-mode MALDI-2 mass spectrometry imaging of cells and tissues at subcellular resolution.Nat. Methods. 2019; 16: 925-931Crossref PubMed Scopus (152) Google Scholar]Sample preparationFluoromethylpyridinium-based reagents facilitating on-tissue covalent charge-tagging of molecules containing phenolic hydroxyls and/or primary or secondary amine groups.Sensitive detection and quantitation of comprehensive neurotransmitter systems of low-abundant neurotransmitters and their associated metabolites (catecholamines and indolamines) and effects of drugs using MALDI- and DESI-MSI. Facilitation of spatial interrogation of neurotransmitter and metabolite pathway alterations in human post-mortem brain tissue and animal models.[33.Shariatgorji M. et al.Comprehensive mapping of neurotransmitter networks by MALDI-MS imaging.Nat. Methods. 2019; 16: 1021-1028Crossref PubMed Scopus (94) Google Scholar,47.Fridjonsdottir E. et al.Mass spectrometry imaging identifies abnormally elevated brain L-DOPA levels and extrastriatal monoaminergic dysregulation in L-DOPA-induced dyskinesia.Sci. Adv. 2021; 7eabe5948Crossref PubMed Scopus (17) Google Scholar,82.Fridjonsdottir E. et al.Region-specific and age-dependent multitarget effects of acetylcholinesterase inhibitor tacrine on comprehensive neurotransmitter systems.ACS Chem. Biol. 2022; 17: 147-158Crossref PubMed Scopus (3) Google Scholar,83.Shariatgorji R. et al.Spatial visualization of comprehensive brain neurotransmitter systems and neuroactive substances by selective in situ chemical derivatization mass spectrometry imaging.Nat. Protoc. 2021; 16: 3298-3321Crossref PubMed Scopus (13) Google Scholar]On-tissue chemical derivatization strategies and instrumental technologies facilitating detection of lipid isomersMapping of molecular species (with tertiary structural information) within cells and tissue sections. Enables studies of drug-related molecular changes in tertiary-structure level and molecular interactions within brain tissue sections.[68.Djambazova K.V. et al.Resolving the complexity of spatial lipidomics using MALDI TIMS imaging mass spectrometry.Anal. Chem. 2020; 92: 13290-13297Crossref PubMed Scopus (35) Google Scholar,84.Wäldchen F. et al.Multifunctional reactive MALDI matrix enabling high-lateral resolution dual polarity MS imaging and lipid C C position-resolved MS2 imaging.Anal. Chem. 2020; 92: 14130-14138Crossref PubMed Scopus (35) Google Scholar,85.Paine M.R.L. et al.Mass spectrometry imaging with isomeric resolution enabled by ozone-induced dissociation.Angew. Chem. Int. Ed. Engl. 2018; 57: 10530-10534Crossref PubMed Scopus (117) Google Scholar]ApplicationsFully cross-validated method according to authorities' guidelines for absolute quantitation analysis of a drug in brain tissue regions using two different MALDI-MSI mass analyzersMALDI-MSI bioanalytical method validation enables its implementation for routine use in pharmaceutical drug development. Imaging of absolute drug concentrations in different brain regions and the effect on drug target(s).[30.Kallback P. et al.Cross-validated matrix-assisted laser desorption/ionization mass spectrometry imaging quantitation protocol for a pharmaceutical drug and its drug-target effects in the brain using time-of-flight and Fourier transform ion cyclotron resonance analyzers.Anal. Chem. 2020; 92: 14676-14684Crossref PubMed Scopus (14) Google Scholar]Imaging the effect of drug–drug interactions on drug BBB permeability. Quantitative imaging of brain distributions of drugs with different BBB permeability directly in brain tissue sections.DESI- and MALDI-MSI enable studies of BBB transport of different drugs and definition of drug distributions in small brain structures. Provide sophisticated spatial information that may reveal unknown drug–target interactions and/or highlight pharmacokinetic properties.[39.Vallianatou T. et al.A mass spectrometry imaging approach for investigating how drug-drug interactions influence drug blood-brain barrier permeability.Neuroimage. 2018; 172: 808-816Crossref PubMed Scopus (24) Google Scholar]Quantitative imaging to determine the extent of unbound drug BBB transport and the post-BBB cerebral distribution of drugs at regional and subregional levels in the brain. An approach that merges in vivo and in vitro neuropharmacokinetic investigations.Differentiation of regional and subregional BBB drug transport characteristics at 20-μm resolution in small brain regions. Allows investigation of heterogeneity in BBB transport. Facilitate interpretation of brain regional target-site drug exposure results and decision-making.[40.Luptakova D. et al.Neuropharmacokinetic visualization of regional and subregional unbound antipsychotic drug transport across the blood-brain barrier.Mol. Psychiatry. 2021; 26: 7732-7745Crossref PubMed Scopus (10) Google Scholar]Integration with other imaging modalities (multiomics) for enhanced molecular understanding of brain functions.Combination with multimodal imaging technologies (e.g., fluorescence, immunofluorescence, mass cytometry) provide novel insights and complementary information to explore molecular, structural and immunological pathways of complex mechanisms in the brain.[71.Kaya I. et al.Brain region-specific amyloid plaque-associated myelin lipid loss, APOE deposition and disruption of the myelin sheath in familial Alzheimer's disease mice.J. Neurochem. 2020; 154: 84-98Crossref PubMed Scopus (33) Google Scholar,86.Kaya I. et al.Histology-compatible MALDI mass spectrometry based imaging of neuronal lipids for subsequent immunofluorescent staining.Anal. Chem. 2017; 89: 4685-4694Crossref PubMed Scopus (47) Google Scholar]Data analysisMachine and deep learning methods and computational analysis of MSI dataPredicting features and patterns in datasets using supervised machine learning algorithms. Exploratory data analysis using unsupervised computational methods.[23.Verbeeck N. et al.Unsupervised machine learning for exploratory data analysis in imaging mass spectrometry.Mass Spectrom. Rev. 2020; 39: 245-291Crossref PubMed Scopus (96) Google Scholar,87.Abdelmoula W.M. et al.Peak learning of mass spectrometry imaging data using artificial neural networks.Nat. Commun. 2021; 12: 5544Crossref PubMed Scopus (24) Google Scholar] Open table in a new tab MALDI-MSI is widely used because it provides high lateral and spatial resolution [8.Kompauer M. et al.Atmospheric pressure MALDI mass spectrometry imaging of tissues and cells at 1.4-μm lateral resolution.Nat. Methods. 2017; 14: 90-96Crossref PubMed Scopus (336) Google Scholar], with fast data acquisition speed (Table 1) [9.Bednarik A. et al.MALDI MS imaging at acquisition rates exceeding 100 pixels per second.J. Am. Soc. Mass Spectrom. 2019; 30: 289-298Crossref PubMed Scopus (15) Google Scholar]. The development of lasers with repetition rates up to 20 kHz [10.Trim P.J. et al.Introduction of a 20 kHz Nd:YVO4 laser into a hybrid quadrupole time-of-flight mass spectrometer for MALDI-MS imaging.Anal. Bioanal. Chem. 2010; 397: 3409-3419Crossref PubMed Scopus (68) Google Scholar] and continuous raster imaging sampling have increased the acquisition rate to up to 100 pixels/s [9.Bednarik A. et al.MALDI MS imaging at acquisition rates exceeding 100 pixels per second.J. Am. Soc. Mass Spectrom. 2019; 30: 289-298Crossref PubMed Scopus (15) Google Scholar]. DESI-MSI is an ambient MS technique that offers higher lateral resolution (up to 25 μm) and sensitivity within the therapeutic drug range [1.Goodwin R.J.A. et al.A critical and concise review of mass spectrometry applied to imaging in drug discovery.SLAS Discov. 2020; 25: 963-976Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar]. Moreover, it does not require complex sample preparation procedures, so it is compatible with traditional histopathological workflows, that is, it enables direct analysis of tissue sections without the use of matrix-coated and conductive glass slides. In nano-DESI, a variant of DESI, the size of the liquid bridge formed between the DESI nebulizer and nanospray capillaries determines the spatial resolution [11.Yin R. et al.High spatial resolution imaging of biological tissues using nanospray desorption electrospray ionization mass spectrometry.Nat. Protoc. 2019; 14: 3445-3470Crossref PubMed Scopus (80) Google Scholar]. SIMS imaging is the technique that currently offers the highest lateral resolution (sub-micrometer) [5.Thomen A. et al.Subcellular mass spectrometry imaging and absolute quantitative analysis across organelles.ACS Nano. 2020; 14: 4316-4325Crossref PubMed Scopus (37) Google Scholar,12.Massonnet P. Heeren R.M.A. A concise tutorial review of TOF-SIMS based molecular and cellular imaging.J. Anal. At. Spectrom. 2019; 34: 2217-2228Crossref Google Scholar]. Due to these (and other) improvements, MSI has become an indispensable tool in drug research. To acquire high-quality MSI data, optimized sample preparation is required to preserve tissue integrity and prevent analyte delocalization [13.Goodwin R.J.A. Sample preparation for mass spectrometry imaging: small mistakes can lead to big consequences.J. Proteome. 2012; 75: 4893-4911Crossref PubMed Scopus (206) Google Scholar]. A critical step in MALDI-MSI sample preparation is matrix application. Previous methods of matrix application involved spotting or electrospraying of an appropriate matrix solution, while recent methods include use of robotic technology, such as acoustic spotters, pneumatic sprayers, and sublimation. These methods of matrix application allow for high sensitivity and lateral resolution, and promote controlled analyte extraction from the tissue surface [3.Norris J.L. Caprioli R.M. Analysis of tissue specimens by matrix-assisted laser desorption/ionization imaging mass spectrometry in biological and clinical research.Chem. Rev. 2013; 113: 2309-2342Crossref PubMed Scopus (505) Google Scholar]. In addition to improved ion sources, such as MALDI laser-induced postionization (Table 1) [14.Niehaus M. et al.Transmission-mode MALDI-2 mass spectrometry imaging of cells and tissues at subcellular resolution.Nat. Methods. 2019; 16: 925-931Crossref PubMed Scopus (152) Google Scholar], mass analyzers with high resolving power and mass accuracy have been developed for small molecule imaging, which allow separation of analytes of interest from background tissue and matrix signals [6.Buchberger A.R. et al.Mass spectrometry imaging: a review of emerging advancements and future insights.Anal. Chem. 2018; 90: 240-265Crossref PubMed Scopus (450) Google Scholar]. Powerful mass analyzers, such as Fourier-transform ion cyclotron resonance (FTICR)-, orbitrap-, and multi-reflecting time-of-flight (MR-ToF) MS instruments, enable high mass resolution (m/Δm up to 1 000 000, where m is the nominal mass/charge ratio for a peak in the mass spectrum, and Δm is peak width at 50% of peak height) and accuracy (<1 ppm) [15.DeLaney K. et al.Advances in high-resolution MALDI mass spectrometry for neurobiology.Mass Spectrom. Rev. 2022; 41: 194-214Crossref PubMed Scopus (9) Google Scholar]. Recently, MALDI-ToF has been combined with ion mobility spectrometry to enhance the separation of molecules in complex biological samples and isomeric and isobaric compounds [16.Ridgeway M.E. et al.Trapped ion mobility spectrometry: a short review.Int. J. Mass Spectrom. 2018; 425: 14Crossref Scopus (169) Google Scholar]. MSI can be easily combined with complementary imaging techniques, so called multimodal imaging, such as traditional histological staining and fluorescent imaging, for the co-registration of analyte distribution in heterogeneous cell populations, thereby paving the way for anatomical, functional, and molecular-level analyses (Table 1) [17.Porta Siegel T. et al.Mass spectrometry imaging and integration with other imaging modalities for greater molecular understanding of biological tissues.Mol. Imaging Biol. 2018; 20: 888-901Crossref PubMed Scopus (94) Google Scholar,18.Vermeulen I. et al.Multimodal molecular imaging in drug discovery and development.Drug Discov. Today. 2022; 27: 2086-2099Crossref PubMed Scopus (6) Google Scholar]. Multimodal imaging enables the correlation of drugs, metabolites, lipids, peptides, or proteins by MSI with histological and/or pathological features and/or tissue substructures, providing highly relevant complimentary information [17.Porta Siegel T. et al.Mass spectrometry imaging and integration with other imaging modalities for greater molecular understanding of biological tissues.Mol. Imaging Biol. 2018; 20: 888-901Crossref PubMed Scopus (94) Google Scholar]. Several examples of combining MSI with other imaging modalities, such as confocal Raman microscopy, imaging mass cytometry, magnetic resonance imaging, and positron emission tomography have been reported [17.Porta Siegel T. et al.Mass spectrometry imaging and integration with other imaging modalities for greater molecular understanding of biological tissues.Mol. Imaging Biol. 2018; 20: 888-901Crossref PubMed Scopus (94) Google Scholar, 18.Vermeulen I. et al.Multimodal molecular imaging in drug discovery and development.Drug Discov. Today. 2022; 27: 2086-2099Crossref PubMed Scopus (6) Google Scholar, 19.Strittmatter N. et al.Holistic characterization of a Salmonella Typhimurium infection model using integrated molecular imaging.J. Am. Soc. Mass Spectrom. 2021; 32: 2791-2802Crossref PubMed Scopus (4) Google Scholar, 20.Henderson F. et al.Multi-modal imaging of long-term recovery post-stroke by positron emission tomography and matrix-assisted laser desorption/ionisation mass spectrometry.Rapid Commun. Mass Spectrom. 2018; 32: 721-729Crossref PubMed Scopus (11) Google Scholar, 21.Piehowski P.D. et al.Automated mass spectrometry imaging of over 2000 proteins from tissue sections at 100-μm spatial resolution.Nat. Commun. 2020; 11: 8Crossref PubMed Scopus (87) Google Scholar]. Complementary application of these modalities, in combination with machine learning, deep learning, and specialized quantitative software [22.Kallback P. et al.Novel mass spectrometry imaging software assisting labeled normalization and quantitation of drugs and neuropeptides directly in tissue sections.J. Proteome. 2012; 75: 4941-4951Crossref PubMed Scopus (115) Google Scholar, 23.Verbeeck N. et al.Unsupervised machine learning for exploratory data analysis in imaging mass spectrometry.Mass Spectrom. Rev. 2020; 39: 245-291Crossref PubMed Scopus (96) Google Scholar, 24.Ovchinnikova K. et al.OffsampleAI: artificial intelligence approach to recognize off-sample mass spectrometry images.BMC Bioinforma. 2020; 21: 129Crossref PubMed Scopus (20) Google Scholar], has revolutionized preclinical drug discovery and greatly improved the significance of data obtained from MSI experiments (Table 1) [25.Liebal U.W. et al.Machine learning applications for mass spectrometry-based metabolomics.Metabolites. 2020; 10: 243Crossref PubMed Scopus (114) Google Scholar,26.Vallianatou T. et al.Integration of mass spectrometry imaging and machine learning visualizes region-specific age-induced and drug-target metabolic perturbations in the brain.ACS Chem. Neurosci. 2021; 12: 1811-1823Crossref PubMed Scopus (10) Google Scholar]. Recently, the imaging of intact biomolecules using MALDI or DESI-MSI has been extended to three dimensions, enabling acquisition of spatial distributions of analytes with depth within volumes of brain tissue specimens. This is usually done by acquiring data on serial consecutive sections of a sample, then stacking and reconstructing the 2D images of each section into a 3D MSI dataset computationally [27.Vos D.R.N. et al.Experimental and data analysis considerations for three-dimensional mass spectrometry imaging in biomedical research.Mol. Imaging Biol. 2021; 23: 149-159Crossref PubMed Scopus (15) Google Scholar]. For instance, 3D MALDI-MSI has been used to visualize the heterogeneous distribution of erlotinib and its related metabolites within brain tissue sections of a patient-derived xenograft mouse model of glioblastoma. The results indicated that the dose level of the drug was higher in the tumor regions than in normal brain parenchyma [28.Randall E.C. et al.Integrated mapping of pharmacokinetics and pharmacodynamics in a patient-derived xenograft model of glioblastoma.Nat. Commun. 2018; 9: 4904Crossref PubMed Scopus (49) Google Scholar], highlighting the potential utility of 3D MALDI-MSI for in-depth neuropharmacodynamics. Despite its many advantages, MSI currently has several challenging limitations. Its application for mapping many analytes is restricted by limitations in sensitivity and dynamic range, hence careful choice of instrument type and optimization of both settings and sample preparation protocols is required to maximize their detectability. Speed of acquisition is a limitation for applications that demand high spatial resolution, although technological developments are continually increasing pixel-to-pixel data collection speeds. MALDI instruments that acquire images at frequencies up to 10 kHz, and hence at up to 40 pixel/s are commercially available. Furthermore, depending on the tissue sample size, image lateral resolution, and mass spectral resolution, individual MSI datasets can contain many hundreds of GB of data in total, which may be challenging for software used for processing and interpreting acquired information. Other limitations of MALDI for certain molecules are due to the laser-induced auto-oxidation of endogenous biomolecules with reducing properties, such as the conversion of glutathione to glutathione sulfate and hypotaurine to taurine [29.Shiota M. et al.Gold-nanofève surface-enhanced Raman spectroscopy visualizes hypotaurine as a robust anti-oxidant consumed in cancer survival.Nat. Commun. 2018; 9: 1561Crossref PubMed Scopus (61) Google Scholar]. In spatial pharmacokinetic studies, MSI experiments enable absolute quantitation of analytes of interest, provided that authorities' guidelines for validation of analytical methods are strictly followed to ensure that the data are reliable and reproducible [30.Kallback P. et al.Cross-validated matrix-assisted laser desorption/ionization mass spectrometry imaging quantitation protocol for a pharmaceutical drug and its drug-target effects in the brain using time-of-flight and Fourier transform ion cyclotron resonance analyzers.Anal. Chem. 2020; 92: 14676-14684Crossref PubMed Scopus (14) Google Scholar]. This involves application of calibration standards to control tissues and spraying tissues with deuterated analogs to use as internal standards for data normalization [22.Kallback P. et al.Novel mass spectrometry imaging software assisting labeled normalization and quantitation of drugs and neuropeptides directly in tissue sections.J. Proteome. 2012; 75: 4941-4951Crossref PubMed Scopus (115) Google Scholar,30.Kallback P. et al.Cross-validated matrix-assisted laser desorption/ionization mass spectrometry imaging quantitation protocol for a pharmaceutical drug and its drug-target effects in the brain using time-of-flight and Fourier transform ion cyclotron resonance analyzers.Anal. Chem. 2020; 92: 14676-14684Crossref PubMed Scopus (14) Google Scholar]. The approach yields similar results to those of liquid chromatography-MS, which is considered the reference method for quantitative studies [30.Kallback P. et al.Cross-validated matrix-assisted laser desorption/ionization mass spectrometry imaging quantitation protocol for a pharmaceutical drug and its drug-target effects in the brain using time-of-flight and Fourier transform ion cyclotron resonance analyzers.Anal. Chem. 2020; 92: 14676-14684Crossref PubMed Scopus (14) Google Scholar]. Therefore, with use of appropriate methods and validation, MSI has comparable utility to established quantitative analytical methods (Table 1). Due to the mentioned advances, MSI is routinely used to monitor drug and drug metabolite concentrations for evaluating drug absorption, distribution, metabolism, excretion, and toxicology (ADMET) in target organs [31.Castellino S. et al.The emergence of imaging mass spectrometry in drug discovery and development: Making a difference by driving decision making.J. Mass Spectrom. 2021; 56e4717Crossref PubMed Scopus (6) Google Scholar]. MSI can significantly expedite preclinical drug development by enabling determination of pharmaceutical agents delivery and distribution in target tissues. MSI has also emerged as an important tool in molecular pathology and surgical fields, allowing for the rapid identification of pathological markers to assist clinical decision [32.Basu S.S. et al.Rapid MALDI mass spectrometry imaging for surgical pathology.NPJ Precis Oncol. 2019; 3: 17Crossref PubMed Scopus (43) Google Scholar]. Despite the obvious advances of MSI there are still challenges associated with pharmacokinetic studies involving MSI, the most significant being the lack of clear guidance for bioanalytical method validation. MSI is a powerful technique for localizing analytes in specific brain regions. Functional MSI i